Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

Natsuka, Shunji; Adachi, Jiro; Kawaguchi, Masahumi; Ichikawa, Akira; Ikura, Koji

doi:10.1271/bbb.66.1174

Cited by 16 publications

(12 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To conduct this, N-glycans were released from membrane protein samples of B4galt5 +/+ -, B4galt5 +/− - and B4galt5 −/− -derived MEF cells by hydrazinolysis, and then pyridylaminated. Then, they were treated with mild acid to convert acidic PA-oligosaccharides to neutral ones [38], and subjected to HPLC using two different columns [33]. Size-fractionation column chromatography using an amide column showed that PA-oligosaccharides from three samples are separated into 11 fractions as indicated below individual peaks in Fig.…”

Section: Resultsmentioning

confidence: 99%

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

et al. 2010

Self Cite

View full text Add to dashboard Cite

Human β-1,4-galactosyltransferase (β-1,4-GalT) V was shown to be involved in the biosynthesis of N-glycans, O-glycans and lactosylceramide (Lac-Cer) by in vitro studies. To determine its substrate specificity, enzymatic activity and its products were analyzed using mouse embryonic fibroblast (MEF) cells from β-1,4-GalT V (B4galt5)-mutant mice. Analysis of expression levels of the β-1,4-GalT I-VI genes revealed that the expression of the β-1,4-GalT V gene in B4galt5+/−- and B4galt5−/−-derived MEF cells are a half and null when compared to that of B4galt5+/+-derived MEF cells without altering the expression levels of other β-1,4-GalT genes. These MEF cells showed no apparent difference in their growth. When β-1,4-GalT activities were determined towards GlcNAcβ-S-pNP, no significant difference in its specific activity was obtained among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. No significant differences were obtained in structures and amounts of N-glycans and lectin bindings to membrane glycoproteins among B4galt5+/+-, B4galt5+/−- and B4galt5−/−-derived MEF cells. However, when cell homogenates were incubated with glucosylcer-amide in the presence of UDP-[3H]Gal, Lac-Cer synthase activity in B4galt5+/−- and B4galt5−/−-derived MEF cells decreased to 41% and 11% of that of B4galt5+/+-derived MEF cells. Consistent with this, amounts of Lac-Cer and its derivative GM3 in B4galt5−/−-derived MEF cells decreased remarkably when compared with those of B4galt5+/+ derived MEF cells. These results indicate that murine β-1,4-GalT V is involved in Lac-Cer biosynthesis.

show abstract

Section: Resultsmentioning

confidence: 99%

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

“…The glycans thus obtained were pyridylaminated using 0.02 ml of coupling reagent and 0.07 ml of freshly prepared reducing reagent as described previously (26,27). The bulk of excess reagents in the reaction mixture was removed by phenol/chloroform extraction (28) and subsequent solid-phase extraction using a Sep-PAK Plus C18 cartridge (29). The pyridylaminated (PA) glycans were analyzed by HPLC.…”

Section: Methodsmentioning

confidence: 99%

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

Hasehira

Tateno

Onuma

et al. 2012

Molecular & Cellular Proteomics

View full text Add to dashboard Cite

We recently reported that induced pluripotent stem cells (iPSCs) prepared from different human origins acquired similar glycan profiles to one another as well as to human embryonic stem cells. Although the results strongly suggested attainment of specific glycan expressions associated with the acquisition of pluripotency, the detailed glycan structures remained to be elucidated. Here, we perform a quantitative glycome analysis targeting both N-and Olinked glycans derived from 201B7 human iPSCs and human dermal fibroblasts as undifferentiated and differentiated cells, respectively. Overall, the fractions of high mannose-type N-linked glycans were significantly increased upon induction of pluripotency. Moreover, it became evident that the type of linkage of Sia on N-linked glycans was dramatically changed from ␣-2-3 to ␣-2-6, and the expression of ␣-1-2 fucose and type 1 LacNAc structures became clearly apparent, while no such glycan epitopes were detected in fibroblasts. The expression profiles of relevant glycosyltransferase genes were fully consistent with these results. These observations indicate unambiguously the manifestation of a "glycome shift" upon conversion to iPSCs, which may not merely be the result of the initialization of gene expression, but could be involved in a more aggressive manner either in the acquisition or maintenance of the undifferentiated state of iPSCs. Molecular

show abstract

“…Another portion (35 l) of the mixture was loaded onto a C 18 cartridge [Presep-C C18(ODS), Wako] to purify the transfer products. After unadsorbed materials were washed out with 3 ml of 0.1% (v/v) acetic acid, the transfer products were eluted with 2 ml of 20% (v/v) methanol in 0.1% acetic acid (Natsuka et al 2002). A mixture of puriWed Gal n -AB was dried in vacuo, re-dissolved in water, and analyzed by MALDI-TOF/MS.…”

Section: Analyses Of Transfer Productsmentioning

confidence: 99%

Chain elongation of pectic β-(1→4)-galactan by a partially purified galactosyltransferase from soybean (Glycine max Merr.) hypocotyls

Konishi

Kotake

Tsumuraya

2007

Planta

View full text Add to dashboard Cite

Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified a beta-(1-->4)-galactosyltransferase (GalT) involved in the synthesis of the beta-(1-->4)-galactan side chains of pectin. The enzyme protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized beta-(1-->4)-galactoheptaose (Gal(7)-AB), leading to the formation of Gal(8-11)-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for 2-aminopyridine-derivatized beta-(1-->4)-galactooligomer acceptors (Konishi et al. in Planta 218:833-842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains to about Gal(35)-AB, thus almost reaching the length (43-47 Gal units) of native beta-(1-->4)-galactan side chains found in pectic polysaccharides from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301-1313, 2002). Enzyme activity increased with increasing chain length of beta-(1-->4)-galactooligomers and reached maximal activity at heptaose, whereas galactooligomers higher than heptaose showed lower acceptor efficiency.

show abstract

Method for Purification of Fluorescence-Labeled Oligosaccharides by Pyridylamination

Cited by 16 publications

References 6 publications

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis

Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells

Chain elongation of pectic β-(1→4)-galactan by a partially purified galactosyltransferase from soybean (Glycine max Merr.) hypocotyls

Contact Info

Product

Resources

About